Spermatogenesis associated serine rich 2 like plays a prognostic factor and therapeutic target in acute myeloid leukemia by regulating the JAK2/STAT3/STAT5 axis

Abstract

Background: Spermatogenesis associated serine rich 2 like (SPATS2L) was highly expressed in homoharringtonine (HHT) resistant acute myeloid leukemia (AML) cell lines. However, its role is little known in AML. The present study aimed to investigate the function of SPATS2L in AML pathogenesis and elucidate the underlying molecular mechanisms.

Methods: Overall survival (OS), event-free survival (EFS), relapse-free survival (RFS) were used to evaluate the prognostic impact of SPATS2L for AML from TCGA database and ourcohort. ShRNA was used to knockdown the expression of SPATS2L. Apoptosis was assessed by flow cytometry. The changes of proteins were assessed by Western blot(WB). A xenotransplantation mice model was used to evaluate in vivo growth and survival. RNA sequencing was performed to elucidate the molecular mechanisms underlying the role of SPATS2L in AML.

Results: SPATS2L expression increased with increasing resistance indexes(RI) in HHT-resistant cell lines we had constructed. Higher SPATS2L expression was observed in intermediate/high-risk patients than in favorable patients. Meanwhile, decreased SPATS2L expression was observed in AML patients achieving complete remission (CR). Multivariate analysis showed high SPATS2L expression was an independent poor predictor of OS, EFS, RFS in AML. SPATS2L knock down (KD) suppressed cell growth, induced apoptosis, and suppressed key proteins of JAK/STAT pathway, such as JAK2, STAT3, STAT5 in AML cells. Inhibiting SPATS2L expression markedly enhanced the pro-apoptotic effects of traditional chemotherapeutics (Ara-c, IDA, and HHT).

Conclusions: High expression of SPATS2L is a poor prognostic factor in AML, and targeting SPATS2L may be a promising therapeutic strategy for AML patients.

Keywords: AML; Apoptosis; Growth; JAK/STAT pathway; Prognostic; SPATS2L; Therapeutic target.

Fig. 1

SPATS2L is upregulated in HHT-resistant cell lines. A Integrative analysis of top 50 up-regulated DEGs of our RNA-seq data from HHT-resistant cell lines to identify DEGs commonly shared among different HHT resistance indexes by the venn method. B The Log₂ fold change of the 29 commonly upregulated DEGs. C The mRNA and protein expression levels of SPATS2L between HHT-sensitive and resistant cells from MV4-11 by qRT-PCR and western blot, respectively. D The mRNA and protein expression levels of SPATS2L between HHT-sensitive and resistant cells from MOLM13 by qRT-PCR and western blot, respectively. NS stands for not significant, P < 0.05 *, P < 0.01 **, P < 0.001 ***

Fig. 2

The prognostic significance of SPATS2L in AML. A OS of SPATS2L low (blue) group and the high (red) group from GEPIA database (log-rank tests; P-value = 0.0059)(http://gepia.cancer-pku.cn/). B OS of SPATS2L low (blue) group and the high (red) group from our cohort (log-rank tests; P-value = 0.009). C EFS of SPATS2L low (blue) group and the high (red) group from our cohort (log-rank tests; P-value < 0.0001). D RFS of SPATS2L low (blue) group and the high (red) group from our cohort (log-rank tests; P-value < 0.0001)

Fig. 3

The regulating function of SPATS2L in AML. A–B. qRT-PCR and western-blot confirmed the decreased level of SPATS2L in AML cells by shRNA targeting KD. C The growth curve of AML cells with or without SPATS2L KD was measured by MTS. D. Flow-cytometry measured the apoptosis of AML cells with or without SPATS2L KD. E. Apoptosis-related proteins were measured by western-blot. NS stands for not significant,, P < 0.05 *, P < 0.01 **, P < 0.001 ***

Fig. 4

The inhibition of SPATS2L in AML can prolong the survival of AML mice. A Schematic illustration of the THP-1 AML xenograft NCG model coupled with control and shRNA targeting SPATS2L KD. B The human CD45 expression of PB and BM cells for the last time in mice was measured by flow cytometry. C The survival curves of the three groups. D Schematic illustration of the THP-1luc AML xenograft NCG model coupled with a control or shRNA targeting SPATS2L KD. E In vivo imaging results of the abdomen of mice on the 7th, 14th, and 21st day after cell injection. E Quantitative analysis results of in vivo imaging results of the abdomen of mice. F Kaplan–Meier curves of the three groups xenotransplanted with human THP-1 luc AML cells. NS not significance, P < 0.05 *, P < 0.01 **, P < 0.001 ***

Fig. 5

The regulating mechanism of SPATS2L in AML. A Integrative analysis of our SPTS2L KD RNA-seq data and TCGA AML RNA-seq data was performed to identify pathways or gene sets commonly enriched by GSEA. Nine gene sets were identified to be affected by both our and TCGA data. B Normalized enrichment scores (NES) of the six gene sets. C Among the nine signaling pathways, IL6-JAK-STAT3-SIGNALING and IL2-STAT5-SIGNALING were significantly suppressed, and the two pathways were most correlated with AML. D The changes of proteins JAK2/STAT3/STAT5 were measured by western-blot

Fig. 6

SPATS2L knockdown enhanced the apoptosis-promoting effect of traditional chemotherapeutics. A IC50 of AML cell lines, THP-1, MV4-11R treated by Ara-c(μM/L), IDA(nM/L), HHT(nM/L). B Protein level of SPATS2L in AML cells treated by Ara-c(μM/L), IDA(nM/L), HHT(nM/L). C Apoptosis of AML cells from THP-cell line among THP-1 scr and THP-1 SPATS2L-sh1, THP-1 SPTS2L-sh2 treated by Ara-c(μM/L), IDA(nM/L), HHT(nM/L), respectively. NS stands for not significant, P < 0.05 *, P < 0.01 **, P < 0.001 ***